1. Field of the Invention
The present invention relates to a method for producing a target substance by using a microorganism, more precisely, means for improving productivity of a substance that is an ultimate target product in a method for producing a target substance such as L-amino acids, antibiotics, vitamins, growth factors and bioactive substances by using a microorganism.
2. Description of the Related Art
As typical methods for producing substances by using microorganisms, there are known methods for producing L-amino acids by fermentation. L-amino acids are used not only as seasonings and foodstuffs but also as components of various nutritional mixtures for medical purposes. Furthermore, they are used as additives for animal feed, reagents in drug manufacturing industry and chemical industry and growth factors for production of L-amino acids such as L-lysine and L-homoserine by use of a microorganism. As microorganisms that can produce L-amino acids by fermentation, there are known coryneform bacteria, Escherichia bacteria, Bacillus bacteria, Serratia bacteria and so forth.
As for such production of target substances by fermentation as described above, it can be said that most of materials used as raw materials are those containing saccharides such as blackstrap molasses. Also in amino acid fermentation or nucleic acid fermentation, culture is performed by using a saccharide as a raw material. Although sugarcane and so forth abundantly contain starch, it is rare to use it as it is as a raw material, but used in most cases as a decomposition product in which starch is decomposed into, for example, monosaccharides or disaccharides. As for the decomposition method, a solution of a saccharifying enzyme such as amylase is generally used, and thereby starch that is polysaccharide is decomposed into relatively low molecular saccharides such as glucose, maltose and maltotriose.
In fermentation using Gram-negative enterobacteria such as Escherichia coli (E. coli), use of such a starch decomposition solution causes a problem. For example, E. coli consumes glucose existing as a main component, but it suffers from the so-called glucose repression, which means that oligosaccharides containing two or more monosacchatides such as maltose are consumed only after monosaccharides are completely consumed. Therefore, if fermentation is terminated when only glucose that is the main component of starch decomposition solution is consumed, oligosaccharides such as maltose are not assimilated but remain vainly. Further, if it is intended to consume oligosaccharides after consumption of glucose, culture time must be extended for that and therefore utility cost and so forth are wasted ineffectively.
It is known that E. coli and Salmonella typhimurium generally suffer from the glucose repression. That is, when it is intended to assimilate glucose together with other carbon sources such as lactose, maltose and glycerol, glucose is assimilated first and the other carbon sources are assimilated later. Monod et al. discovered that, when lactose and glucose were used as carbon sources, two-phase proliferation, i.e., so-called diauxie, was observed (Monod, J., Growth, 11, 223–247, 1947). Through researches in molecular biology, the mechanism thereof is becoming clear. That is, IIAGlc (glucose PTS enzyme II) that acts as a phosphate donor for glucose in the phosphate cascade at the time of assimilation in the glucose-phosphoenolpyruvate-sugar phosphotransferase system, i.e., so-called PTS system, exists in a dephosphorylated state. The dephosphorylated IIAGlc causes the so-called inducer exclusion, in which the dephosphorylated IIAGlc inhibits uptake of the other saccharides (Postma P. W., Lengeler J. W. and Jacobson G. R.: in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt F. C.), pp. 1149–1174, 1996, ASM Press, Washington D.C.).
Uptake of maltose in E. coli suffers form the glucose repression, and this is caused by the interaction between the dephosphorylated IIAGlc and the MalK protein that constitutes the uptake system for maltose by non-PTS. That is, when the bacterium is taking up glucose, IIAGlc excessively exists in the cell, and it binds to the MalK protein to inhibit the uptake of maltose. Further, a mutant strain showing improved uptake of maltose in the presence of glucose analogue was also obtained, and it is known that this mutant strain has a mutation in the malK gene coding for the MalK protein (Dean D. A. et al., Regulation of the Maltose Transport System of Escherichia coli by the Glucose-specific Enzyme III of the Phosphoenolpyruvate-Sugar Phosphotransferase System., J. Biol. Chem., 265 (34), 21005–21010, 1990; Kuhnau, S. et al., The Activities of the Escherichia coli MalK Protein in Maltose Transport and Regulation, and Inducer Exclusion Can Be Separated by Mutations, J. Bacteriol., 173 (7), 2180–2186, 1991).
Further, also for IIAGlc, there was reported a mutant strain that contained a mutant protein showing reduced binding with lactose permease (Zeng, G. A. et al., Mutation alanalysis of the enzyme IIIGlc of the phosphoenolpyruvate phosphotransferease system in Escherichia coli, Res. Microbiol., 143, 251–261, 1992). The lactose permease is an uptake enzyme for lactose that is one of the non-PTS saccharides.
However, it is unknown whether the aforementioned mutant strains assimilate maltose simultaneously in the presence of glucose.